PROJECT SUMMARY Understanding the genome-wide distribution and organization of various epigenetic marks in the context of disease progression or in response to therapeutic intervention has broad applications for both basic and translational research. EpiCypher has developed the first quantitative ChIP approach (R44HG008907), which uses post-translationally modified (PTM)-defined recombinant nucleosomes as internally calibrated ?spike in? standards (ICe-ChIP). This will enable a marked expansion of ChIP applications into new markets including drug discovery and diagnostics, though its full potential is restrained by two current limitations in detection reagents. First, many so-called ?ChIP-grade? antibodies exhibit extensive cross-reactivity with structurally similar PTMs (e.g., trimethylation vs. dimethylation at H3K4 [H3K4me3 vs. H3K4me2]). Second, antibody binding is highly influenced by the presence of neighboring PTMs. Antibodies targeting H3K4me3, for instance, exhibit nearly 2-fold greater binding affinity if phosphorylation is also present at nearby residues. These specificity limitations result from the crude selection strategies by which antibodies are developed. It is therefore, imperative to develop next-generation detection reagents that can have their binding preferences selected for (or tuned) using rigorous and efficient selection strategies. Here, EpiCypher will develop RNA aptamers as highly specific detection reagents for ChIP applications. Aptamers are synthetically derived using an in vitro directed evolution approach (SELEX: Systematic Evolution of Ligands by EXponential enrichment), thereby mitigating animal involvement and allowing specific binding preferences to be positively and negatively selected a priori. The ability to finely tune aptamer binding affinity and specificity without the expenditure of animal life provides a marked advantage over antibody-based reagents. Recent advances in aptamer technology have enabled these reagents to be highly resistant to degradation in cell lysates (a feature required for ChIP applications). However their development towards histone PTMs has been restricted by the highly charged nature of chromatin. To meet this challenge, EpiCypher is collaborating with Dr. Matthew Levy to develop a novel engineering strategy to generate aptamers that can tolerate such highly charged target motifs. The innovation of this project is a unique selection strategy and the incorporation of hydrophobic residues to generate aptamers that can bind PTMs on charged histones with exquisite target specificity. In Aim 1, we will establish an optimization pipeline to develop aptamers specific for histone H3K4 methylation and validate their binding specificity using EpiCypher's combinatorially-modified histone peptide array (EpiTitanTM). In Aim 2, we will demonstrate the utility of our novel aptamers in ICe-ChAP (Chromatin AptamerPrecipitation) and compare these results with best-in-class antibodies capable of distinguishing various H3K4 methylation states. In Phase II, we will scale- up commercial production of aptamers that target H3K4 methylation and other disease-relevant PTMs.